Rickettsiae with the exception of Rochalimaea are obligate intracellular parasites. As such, these microorganisms are often difficult to grow and the diseases they cause are difficult to diagnose. Because of the difficulty of growing these microorganisms, large scale preparation of vaccines and antigens has been costly and sometimes impossible. Many of the rickettsiae are the pathogenic agents of vector-transmitted diseases of human and animals. They are transmitted by invertebrate vectors such as ticks. Within rickettsiae, the microorganisms of the Anaplasma, Ehrlichia and Rickettsia genera are the causative agents of vector transmitted diseases and are difficult to grow, especially on a large scale.
Anaplasmosis is the only major tick-borne disease of cattle endemic in the United States. The causative agent, Anaplasma marginale, invades and multiplies in erythrocytes of cattle causing mild to severe anemia. Annual mortality and morbidity due to anaplasmosis among U.S. beef cattle has been estimated at 50,000 to 100,000 head and a cost of $300 million. The average weight loss during acute infections is 86 kg, with increased veterinary costs estimated at $52/head (Palmer in Veterinary Protozoan and Hemoparasite Vaccines, J. G. Wright (Ed.) CRC Press Inc., Boca Raton, Fla. 1989). The resultant loss of cattle for the export market has been estimated at $45 million annually (McCallon 1973).
Anaplasmosis can be transmitted biologically by several species of hard ticks (Dikmans Amer. J. Vet. Res. 38:5 (1950); Ewing, S. Transmission of Anaplasma marginale in Arthropods in Proc: 7th National Anaplasmosis Conference, Mississippi State MS p 395, 1981) or mechanically by blood-contaminated mouthparts of biting flies or fomites. In some geographical areas, transmission has been thought to occur primarily by biting flies and thus, the disease has been called "fly fever". However, ticks most likely play a major role in transmission of this disease. Male ticks are believed to contribute markedly to transmission of A. marginale. (Coan and Stiller, Abst. 70th Am. Pacific Branch Mtg. of Entomological Soc. Amer., Sand Diego, Calif. p.66 (1986); Coan et al., Ann. Western Conf. of Food Ani. Vet. Med., Boise, ID p.34 (1987); Coan et al., Proc. 8th National Vet. Hemoparasit. Dis. Conference, St. Louis, Mo. pp. 1161 (1989); Kocan et al., Am. J. Vet. Res. 53:499 (1992a); Kocan et al., J. Med. Entomol. 29:657 (1992b)). Male ticks can acquire infection as adults by feeding for short periods on infected cattle. Because male ticks are intermittent feeders, they readily detach and can reattach to other cattle during their life. Interhost transfer of male Dermacentor ticks among cattle has been clearly demonstrated. These male ticks have been shown to be persistently infected with A. marginale and are able to transmit the rickettsia repeatedly to susceptible cattle (Kocan et al. 1992b cited supra).
The widening distribution of anaplasmosis is probably contributed to by increased movement of cattle and the lack of sensitivity of the complement-fixation test. The complement-fixation test for anaplasmosis has been shown to lack sensitivity, especially in calves, carrier cattle and in cattle that have been treated with tetracyclines which lowers antibody titers often to undetectable levels (Goff et al., Vet. Microbiol. 24:381 (1990)). Thus, cattle may be shipped as serologically negative when they actually are active carriers of the organism. These carrier cattle are then likely to serve as reservoirs of infection for mechanical transmission by blood-contaminated fomites, biting flies or biologically by ticks.
Serologic diagnosis of anaplasmosis is predominantly accomplished by the complement-fixation test (USDA, A microtiter technique for complement fixation text for anaplasmosis, Manual pp. 1-16, USDA Animal and Plant Health Inspection Service, Beltsville, Md. (1968)). Many other tests have been developed to measure Anaplasma-specific antibodies, including the capillary-tube agglutination test (Ristic, J. Am. Vet. Assoc. 141:588 (1962)), card test (Amerault & Roby, J. Am. Vet. Assoc. 153:1828 (1968)), latex agglutination (Montenegro-James et al., Vet. Parasitology 8:241 (1981)), indirect fluorescent antibody test (IFAT) (Goff et al. 1990, cited supra), conventional ELISA (Shkap et al., Vet. Microb. 25:45 (1990)) and radioimmunoassay (Schunter & Leatch, Am. J. Vet. Res. 49:504 (1988)). Growth of A. marginale in cell culture may provide an inexpensive and more consistent antigen for use in serologic tests.
Control strategies for anaplasmosis in Oklahoma and elsewhere varies owing to the complexity of the disease and mode of transmission, and may include vector control, administration of tetracyclines, vaccination and maintenance of uninfected cattle. Current vaccines marketed in the U.S. are killed vaccines made from infected bovine blood. These vaccines reduce clinical symptoms but do not prevent infection. Vaccinated cattle that are challenge-exposed thereafter become carriers and their blood can serve as an infective source for ticks and/or mechanical transmission via biting flies and blood-contaminated instruments. Subunit vaccines which have been described provide only partial protection against challenge (Vidotto et al., Inf. and Imm. 62:2940 (1994)). In addition, many vaccine preparations can be contaminated with bovine red blood cell antigens as well as with other bovine pathogens.
Numerous attempts to grow A. marginale in a variety of mammalian and invertebrate cells have failed. Attempts included cell lines from D. variabilis ticks but none resulted in a continuous culture system capable of producing cattle-infective rickettsiae. A. marginale apparently does not invade nucleated cells in its vertebrate host, only erythrocytes. In contrast, the parasite develops in several types of nucleated tick cells (in midgut, gut muscle and salivary glands).
Erlichieae are rickettsial pathogens closely related to Anaplasma. Those species for which a biological vector is known are transmitted by ticks. While Anaplasma is confined to red cells in its mammalian host, the tick-borne Ehrlichieae infect white blood cells. Typically, Anaplasma and Ehrlichia are contained within membrane-bound vacuoles of their respective host cells.
The monocytic Ehrlichia species include Ehrlichia canis of dogs and Ehrlichia chaffeensis, E. risticii and E. sennetsu. These microorganisms are genetically related and cross-react serologically. The first Ehrlichia species recognized was E. canis. It occurs in all areas of the world where the vector tick, Rhipicephalus sanguineus (the brown dog tick), lives. The disease it causes is sometimes called canine tropical pancytopenia. It is a problem especially in all warm areas of the world, e.g., the southern U.S., Central and South America, the Mediterranean, South Asia (Ristic, M. and Huxsoll, D. L., 1984); Tribe II. Ehrlichieae Philip 1957. In: Bergey's manual of Systematic Bacteriology; Vol. 1, section 9). Ehrlichia canis can be cultivated in the dog cell line DH82 as well as human-dog hybrid cell lines (See: Rikihisa, Y. 1991. Clinical Microbiology Reviews, 4:286). Ehrlichia chaffeensis has been recognized only recently, and is associated with human ehrlichiosis. Maeda, K. et al., 1987, N. Eng. J. Med. 316:853; Dawson, J. E. et al., 1991, J. Clin. Microbiol. 29:2741). E. chaffeensis can also be cultured in DH82 cells. E. risticii is the causative agent of Potomac horse fever. This disease is known to occur in North America, France and India. E. risticii can be grown in macrophage-monocyte cell lines such as P388D.sub.1, T-84 and U937. E. sennetsu is the causative agent of Sennetsu erlichiosis of humans. E. sennetsu grows in mice and cell lines such P388D.sub.1 cells, L929 cells, and Hela cells.
Infections with monocytic Ehrlichiae can be diagnosed by direct microscopic examination and/or serodiagnosis. Treatment with antibiotics is effective. Two vaccines prepared from inactivated cell cultured E. risticii are commercially available for Potomac Horse Fever but none have been developed for large scale prevention of other ehrlichial diseases.
Rickettsia rickettsii is the causative agent of Rocky Mountain spotted fever. In the western U.S., the main vector of this human pathogen is the Rocky Mountain wood tick, Dermacentor andersoni. A close relative, Dermacentor variabilis also known as the American dog tick or common wood tick, is responsible for transmission in the eastern parts of the country. The disease is characterized by an acute febrile phase, typically with a disseminated rash. Unlike Anaplasma and Ehrlichia, which are passed only transstadially in ticks (from larvae to nymphs and/or nymphs to adults), R. rickettsii is transmitted transovarially and transstadially in the vector. After entry into the host cell, spotted fever rickettsiae do not remain in the phagosome, but quickly escape into the host cytoplasm, and commonly invade the nucleus. Their ability to move quickly from cell to cell by making use of the host cell's cytoskeletal elements is a characteristic feature leading to early dissemination to multiple tissues.
Vaccines against Rocky Mountain spotted fever were produced from inactivated organisms grown in embryonated hen's eggs, and more recently, in mammalian cell cultures. These vaccines are capable of preventing clinical illness, but not necessarily infection. Broad spectrum antibiotics, particularly chloramphenicol and tetracyclines, are highly effective when used promptly.
Thus there is a need to develop a culture system for growing rickettsiae in tick cell culture for use in diagnostics, and preparation of antigens and vaccines. There is a need to develop vaccines and antigen preparations that effectively protect against infection with Anaplasma marginale but that are not contaminated with bovine red blood cells or bovine pathogens. There is a need to develop a large scale culture system for preparation of large amounts of antigen from rickettsiae such as Anaplasma marginale and Ehrlichia canis for use in diagnostics and vaccines.